1. Field of the Invention
This invention relates to the development of transgenic plum genotypes which flower very early and continually and produce normal fruits and fertile seeds within six to twelve months and the seeds and plants obtained from such transgenic plants. The invention also relates to a method of transforming plum plant cells and plum plants utilizing a recombinant vector containing the construct comprising the gene for early flowering, PtFT1.
2. Description of the Relevant Art
Prunus is the horticulturally valuable genus in the family Rosaceae. Members of the family Rosaceae are cultivated for their fruits (peaches, plums, apricots, nectarines, cherries), nuts (almond) or for their ornamental flowers (flowering cherries). In addition to being a dietary supplement important for human health, Prunus fruits are a rich source of antioxidants which are widely reported to reduce cancer risks in humans. Conventional breeding and the application of molecular genetic technology such as structural and functional genomics and genetic engineering can be used to improve Prunus species. Currently, numerous genes in rosaceous fruit trees and related species have been identified through world-wide efforts of genome analyses (Retrieved from the Internet: <URL: bioinform.wsu.edu/gdr), but characterization of gene function through overexpression or gene silencing approaches in transgenic plants for genetic improvement of Prunus fruit trees is still problematic (Shulaev et al. 2008. Plant Physiol. 147: 985-1003).
Fruit tree breeding is a slow, arduous process that has changed little over the centuries. The long juvenile (pre-flowering) period of three to eight years is a severe impediment to the genetic improvement of both conventionally bred and transgenic Prunus fruit trees. Several generations of backcrossing and selection are required to develop improved Prunus cultivars; this process normally takes more than 20 years (Scorza, R. 2001. HortScience 36: 855-858). Limitations also include large land areas with significant field costs, and yearly limitations on flowering and fruiting related to chill and heat requirements. Shortening the 3-8 year juvenile period of Prunus fruit trees to a year or less by inducing early flowering in fruit trees could dramatically reduce the time, space and cost required for genetic improvement of fruit trees and result in the production of better quality fruits.
Tree fruits are temperate crops which are cultivated in orchard systems and require a period of chilling for continued growth and fruit production. They produce a single crop of fruit/nuts per year, the timing of which depends on the species and variety but is typically between the months of June and September. The amount of chill needed for each variety and the timing of flowering and fruit set impose significant barriers to where individual species and cultivars can be productively grown. This situation results in product surplus during summer months and a lack of product in the winter months but often filled by foreign imports. The ability to alter these crops such that they are no longer limited by time of chilling and/or extend the production season through continued flowering and fruit set would provide substantial improvements to crop productivity and market delivery.
Molecular genetics of flowering has been widely reported in the model plant and ectopically overexpressed flower-inducing genes have produced early flowering in Arabidopsis and other herbaceous plants. Through many studies in Arabidopsis, the pathway to flower determination has been resolved. Normally, there is an interaction between temperature and light which affects the levels of FLOWERING LOCUS C (FLC) and CONSTANS (CO), respectively. FLC negatively regulates FLOWERING LOCUS T (FT) and CO positively regulates FT. FT induces APETALA1 (AP1), FRUITFUL (FUL) and SUPPRESSOR OF CONSTANS OVEREXPRESSION 1 (SOC1), and SOC1 induces LEAFY (LFY). Other genes associated with flower induction include: FLOWERING LOCUS D (FD), and CAULIFLOWER (CAL). Overexpression of these genes individually or collectively and the silencing of TERMINAL FLOWER 1 (TFL1), can induce early flowering in Arabidopsis and other plants; for review, see Parcy, F. (2005. Int. J. Dev. Biol. 49: 585-593). LFY and AP1 determine flower meristems (Parcy, supra); AP1, LFY, FT and FUL have been used to shorten the juvenility period in trees resulting in early flowering. Several MADS-box genes also induce early flowering in plants. Since flowering is an essential process for the survival of plant species, floral-related gene redundancy is common. FT is now considered as the elusive flowering signal ‘florigen’. Based on the current knowledge, it appears that FT protein produced in companion cells of phloem in small veins of leaves is translocated to shoot apical meristems where it activates the shoot apical meristem-specific transcription factor FD which in turn recruits the meristem-identity gene LFY and its homologs AP1 and CAL to induce flowering in plants (Abe et al. 2005. Science 309: 1052-1056; Parcy, supra; Wigge et al. 2005. Science 309: 1056-1059).
Several flower-inducing genes discovered in Arabidopsis have been tested in woody perennial plant systems. Some form of early flowering has been reported in apple, citrus, and poplar by overexpressing MADS4 and FT genes. Flachowsky et al. (2007a. Acta Hort. 738: 307-312) obtained an early flowering clone of the apple cultivar ‘Pinova’ by over expressing a silver birch (Betula pendula Roth) floral meristem identity MADS-box gene, bpMADS4. These transgenic apple plants flowered within 13 weeks of transformation and initially produced solitary flowers, but later produced clusters of 5 flowers. Pollination of these flowers with Malus fusca pollen produced normal fruits and seeds (Flachowsky et al. 2007b. Plant Breeding 126: 137-145). In citrus, constitutive expression of citrus FT (CiFT) in trifoliated orange (Poncirus trifoliate) induced early flowering as early as 12 weeks after transfer of transgenic plants to a greenhouse (Endo et al. 2005. Trans Res. 14: 703-712). The transgenic lines showed variation in phenotypes such as time of first flowering and tree shape. Two FT genes have been isolated from poplar. PtFT1 has been isolated from Populus trichocarpa (Bohlenius et al. 2006. Science 312: 1040-1043) and PtFT2 is from P. deltoides (Hsu et al. 2006. Plant Cell 18: 1846-1861). Male poplar hybrid P. tremula×P. tremuloides flowered as early as four weeks post-transformation when overexpressing PtFT1, whereas PtFT2, which has 91% similarity in coding regions at the amino acid level, induced flowering after a year. It appears that PtFT2 is involved in seasonal flowering. Although early flowering has been achieved by overexpressing several MADS-box genes and transcription factors, these genes are multifunctional and overexpression of these genes induced alterations both in vegetative and reproductive growth and development. However, FT is neither a transcription factor nor a MADS-box gene and overexpression of FT did not adversely affect normal growth and development of plants (Bohlenius, supra).
The tree fruit industry is facing challenges of climate change, reductions in available labor, the need for reduced chemical inputs, and the spread of exotic pests and pathogens. To meet these challenges the development of improved varieties is vital. The objective of our research was to utilize the knowledge of the molecular genetics of flowering gained in Arabidopsis and to design and implement a strategy and model system to routinely induce early flowering and normal fruiting in Prunus. 